Project Summary Co-infection with HBV and HIV-1 is common, and HIV co-infection can exacerbate progression of viral hepatitis and accelerate liver disease progression. A major hurdle to elucidating potential mechanisms underlying these common and serious diseases, and thus to developing effective therapies, is the lack of small animal models that reproduce human-like infection with HBV and HIV-1. Both viruses exhibit a narrow host range in which infection is largely limited to humans and other great apes. Our labs (Ploss and Su) have developed novel humanized mice that are co-engrafted with human livers and human immune cells (FNRGF/A2-hu HEP/HSC mice), with human hepatocytes and improved human myeloid cell/DC and NK cell subsets that are underrepresented in conventional humanized mouse models. We have demonstrated that these refinements yield vaccine-induced immunogenicity profiles resembling those of humans who have been vaccinated with the same well-defined YFV-17D vaccine. We will capitalize on these promising breakthroughs to investigate the following: 1) We will infect cohorts of FNRGF/A2- hu mice dually engrafted with human HLA-matched hepatocytes and human hematopoietic cells with HBV and/or HIV-1. We will monitor, and quantify longitudinally, HBV and HIV viremia and immune activation, including antigen- specific CD8+ T cells and liver disease progression (Aim 1A). We will also perform analyses in cohorts of HBV/HIV co-infected mice which have been treated with HAART (including HBV polymerase inhibitors), to suppress HIV-1 and HBV, to model accurately clinically relevant situations (Aim 1B). 2) We will study HIV/HBV-induced T cell impairment. We will monitor T cell exhaustion markers on HIV and HBV specific T cells and profile functional impairments in antigen specific CD8+ T cells during HBV/HIV co-infections using single-cell transcriptomics (Aim 2A). We will also evaluate the impact of blocking INFAR1 on HIV/HBV-induced immune impairment. We will monitor reversion of T cell exhaustion functions of HIV- and HBV-specific T cells and profile IFN-induced functional impairments in antigen specific CD8+ T cells during HBV/HIV co-infections (Aim 2B). 3) We will investigate how HIV-1 infection elevates HBV-induced pathogenic macrophages in the liver of humanized mice in vivo (Aim 3A). We will also study how HIV and HBV interact with M2-like macrophages to activate human stellate cells (Aim 3B). Finally, we will study how inhibiting M2-like macrophages prevents or reverses HIV/HBV-induced liver diseases in humanized mice (Aim 3C). We will thus capitalize on our extensive complimentary expertise in virology and immunology of HIV-1/HBV (Su), hepatitis viruses (Ploss) and in humanized mouse technology to achieve the exciting aims. Our work will advance the field of HBV and HIV-1 research by showing that a novel small animal model can be successfully used to understand HIV/HBV coinfection and immune responses, and to model treatments for the associated liver diseases.